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E. coli Plasmid Vectors pp 203–207Cite as

Site-Directed Mutagenesis Using the Megaprimer Method

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Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 235))

Abstract

Site-directed mutagenesis (SDM) is used to introduce a defined mutation into target DNA of known sequence to study, for example, gene expression or protein structure–function relationship. A number of polymerase chain reaction (PCR)-based mutagenesis methods have been developed (1). Among them, the “megaprimer” method is probably the simplest and most flexible (2–5). The megaprimer method utilizes two external oligonucleotide primers and one internal mutagenic primer in two rounds of PCR with a DNA template that contains the sequence to be mutated. The first round of PCR is carried out using one of the external primers and the mutagenic primer containing the desired mutation. This amplifies an intermediate PCR product that is purified and used as a “megaprimer” for the second round of PCR, along with the other external primer. The final PCR product is cloned into appropriate vectors and used in downstream applications.

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Author information

Authors and Affiliations

  1. Department of Surgery, Comprehensive Cancer Center, University of California, San Francisco, CA

    Zhidong Xu

  2. University of Rome and CSS-Mendel Institute, Rome, Italy

    Alessia Colosimo

  3. Department of Medicine, University of Vermont, Colchester, VT

    Dieter C. Gruenert

Authors
  1. Zhidong Xu
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  2. Alessia Colosimo
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  3. Dieter C. Gruenert
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Editor information

Editors and Affiliations

  1. University of California at Berkeley, School of Public Health, Berkeley, CA

    Nicola Casali

  2. Department of Clinical Veterinary Medicine, University of Cambridge, Cambridge, UK

    Andrew Preston

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© 2003 Humana Press Inc.

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Xu, Z., Colosimo, A., Gruenert, D.C. (2003). Site-Directed Mutagenesis Using the Megaprimer Method. In: Casali, N., Preston, A. (eds) E. coli Plasmid Vectors. Methods in Molecular Biology™, vol 235. Humana Press. https://doi.org/10.1385/1-59259-409-3:203

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  • DOI: https://doi.org/10.1385/1-59259-409-3:203

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-151-6

  • Online ISBN: 978-1-59259-409-2

  • eBook Packages: Springer Protocols

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